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Free Zinc Ions Myd88 and TRIF Signaling Pathways by Differential Differential Regulation of TLR-Dependent MyD88 and TRIF Signaling Pathways by Free Zinc Ions This information is current as Anne Brieger, Lothar Rink and Hajo Haase of October 6, 2021. J Immunol 2013; 191:1808-1817; Prepublished online 17 July 2013; doi: 10.4049/jimmunol.1301261 http://www.jimmunol.org/content/191/4/1808 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/07/12/jimmunol.130126 Material 1.DC1 References This article cites 48 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/191/4/1808.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 6, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Differential Regulation of TLR-Dependent MyD88 and TRIF Signaling Pathways by Free Zinc Ions Anne Brieger, Lothar Rink, and Hajo Haase Zinc signals are utilized by several immune cell receptors. One is TLR4, which causes an increase of free zinc ions (Zn2+) that is required for the MyD88-dependent expression of inflammatory cytokines. This study investigates the role of Zn2+ on Toll/IL-1R domain–containing adapter inducing IFN-b (TRIF)–dependent signals, the other major intracellular pathway activated by TLR4. Chelation of Zn2+ with the membrane-permeable chelator N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine augmented TLR4-mediated production of IFN-b and subsequent synthesis of inducible NO synthase and production of NO. The effect is based on Zn2+ acting as a negative regulator of the TRIF pathway via reducing IFN regulatory factor 3 activation. This was also observed with TLR3, the only TLR that signals exclusively via TRIF, but not MyD88, and does not trigger a zinc signal. In contrast, IFN-g–induced NO production was unaffected by N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine. Taken together, Zn2+ is specifically involved in TLR signaling, where it differentially regulates MyD88 and TRIF signaling via a zinc signal or via Downloaded from basal Zn2+ levels, respectively. The Journal of Immunology, 2013, 191: 1808–1817. he essential trace element zinc is indispensable for the NF-kB activation, and thereby for transcription and release of function of the immune system. Zinc deficiency leads to inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 (7). T increased susceptibility to infections, and restoring zinc After activation of the MyD88-dependent pathway, the receptor levels of marginally deficient elderly by supplementation can im- complex is endocytosed (10) and the adaptors TRAM and Toll/IL- http://www.jimmunol.org/ prove immune function (1, 2). In recent years, it has become clear 1R domain–containing adapter inducing IFN-b (TRIF) bind to that the importance of zinc for immunity is based, at least in part, TLR4, resulting in a delayed NF-kB signal and activation of the on a function in signal transduction in cells of the immune system IFN regulatory factor 3 (IRF3) (11). Signaling via TRIF and IRF3 (3). Intracellular zinc concentrations are tightly regulated by zinc triggers the secretion of IFN-b (12). IFN-b binds to the type 1 transporters and zinc signals (4). The latter consists of a change in IFNR, which, in turn, activates a JAK-STAT pathway inducing the the cytoplasmic concentration of free zinc ions (Zn2+), which have expression of surface molecules required for the interaction with already been observed in response to several stimuli and in many T cells, such as CD40, CD80, and CD86 (13, 14). Together, the ac- different types of immune cells. In monocytes, MCP-1 (5), PMA tivation of NF-kB and JAK-STAT also induces the expression of (6), TNF-a, insulin, and the TLR ligands Pam3CSK4 and LPS (7) inducible nitric monoxide synthase (iNOS) (15–17). iNOS activity by guest on October 6, 2021 induce an intracellular increase of the cytosolic free Zn2+ con- is mainly regulated through gene expression, and its product NO is centration. an important mediator of inflammation (18). Functions of iNOS- TLRs are a subgroup of the pattern-recognition receptors, which derived NO are the elimination of microbes, parasites, and cancer detect conserved molecular structures of pathogens that are used as cells (18, 19). danger signals by certain immune cells (8). Ligand binding to the Still, it is poorly understood how the TLR-induced inflammatory ectodomain of TLRs induces dimerization, bringing their cyto- and antiviral immune responses are balanced. Zinc signals affect plasmic Toll/IL-1R domains together, resulting in the recruitment the TLR4-induced production of MyD88-dependent inflammatory of intracellular adapter proteins and initiation of downstream cytokines (7), and the aim of this study is to investigate whether signaling events (9). TLR4 initially binds the adaptors TIRAP and intracellular Zn2+ levels also affect the TLR4-induced activation MyD88, and causes phosphorylation of MAPKs and early acti- of the TRIF pathway, in particular, the release of the inflammatory vation of NF-kB. The zinc signal is required for preventing de- mediator NO. phosphorylation of MAPKs p38, MEK1/2, and ERK1/2, and for Materials and Methods Cell culture Institute of Immunology, Medical Faculty, RWTH Aachen University, 52074 Aachen, All cell lines were cultured at 37˚C in a humidified 5% CO2 atmosphere. Germany RAW 264.7 murine macrophages, L929 fibroblasts, and 7FD3 hybridoma Received for publication May 13, 2013. Accepted for publication June 13, 2013. cells were grown in RPMI 1640 (Lonza, Verviers, Belgium) containing This work was supported by German Research Council (Deutsche Forschungsge- 10% FCS (heat inactivated for 30 min at 56˚C; PAA, Co¨lbe, Germany), 2 meinschaft) Grant HA4318/3 (to H.H.). mM L-glutamine, 100 U/ml potassium penicillin, and 100 U/ml strepto- mycin sulfate (all from Lonza). 3T3 and 293T cells were grown in DMEM Address correspondence and reprint requests to Prof. Hajo Haase, Institute of Immu- nology, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aa- (Lonza) supplemented as described earlier. Zinc-deficient medium was chen, Germany. E-mail address: [email protected] obtained by treatment with CHELEX 100 ion exchange resin (Sigma- Aldrich, St. Louis, MO) as described previously (20). The online version of this article contains supplemental material. Abbreviations used in this article: BMM, bone marrow–derived macrophage; iNOS, Bone marrow–derived macrophages inducible nitric monoxide synthase; IRF3, IFN regulatory factor 3; PTP, protein tyrosine phosphatase; TPEN, N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine; Bone marrow was isolated from the femurs of C57BL/6 mice. A total of 5 TRIF, Toll/IL-1R domain–containing adapter inducing IFN-b. 3 3 10 cells/ml was seeded in 20 ml RPMI 1640 containing 18% heat- inactivated FCS, 2 mM L-glutamine, 100 U/ml potassium penicillin, 100 Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 U/ml streptomycin sulfate, 1 mM nonessential amino acids (Lonza), and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301261 The Journal of Immunology 1809 20% sterile-filtered L929 cell culture supernatant for a total of 8 d. On LumiGlo Reagent (Cell Signaling Technology) on an LAS-3000 (Fujifilm day 4, 10 ml fresh culture medium was added. On day 7, the medium was Lifescience, Du¨sseldorf, Germany). Densitometric quantification was per- completely replaced. On the next day, experiments were performed. formed with Adobe Photoshop Elements. Production of IFN-b–containing cell-free culture supernatant Nuclear extraction for Western blot samples The supernatants of 3T3 and 293T cells stably transfected to produce mouse A total of 5 3 106 cells was thoroughly suspended in 2 ml ice-cold buffer IFN-b were collected 4 d after seeding and were centrifuged for 10 min at (10 mM Tris-HCl, pH 7.4, containing 15 mM NaCl, 60 mM KCl, 0.5% 300 3 g. Supernatants were sterile-filtered with a 0.22-mm filter (Millex- Igepal CA-630, 1 mM EDTA, 0.1 mM EGTA) and lysed at 4˚C for 5 min. GV; Millipore, Billerica, MA), and aliquots were stored at 280˚C until Nuclei were pelleted by centrifugation (600 3 g, 4˚C, 5 min), taken up in use. The activities of the IFN-b–containing cell supernatants were deter- 100-ml Western blot sample buffer, and treated as described earlier for mined by an IFN-b bioassay with the L929 cell line and the Encephalo- Western blot samples. myocarditis virus. For all experiments, 2000 U/ml IFN-b was applied. RT-PCR Purification of monoclonal anti–IFN-b Abs The mRNA of 5 3 105 cells was isolated after lysis in 1 ml Tri Reagent 7FD3 hybridoma cells produce rat IgG1 anti-mouse IFN-b Abs. The cells (Ambion, Life Technologies, Carlsbad, CA) and transcribed into cDNA were grown for 3–4 d under normal culture conditions until confluency and with the qScript cDNA Synthesis Kit (Quanta Biosciences, Darmstadt, cultured for 3 more days in serum-free medium (21). Supernatants were Germany) according to the manufacturers’ instructions. Quantitative real- collected, sterile-filtered, and concentrated (Minicon B15 clinical sample time PCR was performed on an ABI PRISM 7000 or a Step-One plus Real- concentrator; Millipore).
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